Process for producing HMG-CoA reductase inhibitor

ABSTRACT

The present invention relates to a protein derived from a microorganism belonging to the genus  Bacillus , which has an activity of hydroxylating a compound represented by the formula (I-a): 
                         
wherein R 1  represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal, and R 2  represents a substituted or unsubstituted alkyl or a substituted or unsubstituted aryl,
 
or a ring-closed lactone form thereof;
 
a DNA encoding the protein; and a recombinant DNA comprising the DNA.

TECHNICAL FIELD

The present invention relates to a DNA which is related to theproduction of a compound which inhibits hydroxymethylglutaryl CoA(HMG-CoA) reductase and has an action of reducing serum cholesterol, anda process for producing said compound using the DNA.

BACKGROUND ART

A compound represented by the formula (VI-a) (hereinafter referred to ascompound (VI-a)):

wherein R¹ represents a hydrogen atom, a substituted or unsubstitutedalkyl, or an alkali metal; ora lactone form of compound (VI-a) represented by the formula (VI-b)(hereinafter referred to as compound (VI-b)):

is known to inhibit HMG-CoA reductase and exhibit an action of reducingserum cholesterol (The Journal of Antibiotics, 29, 1346 (1976)).

There have been several reports regarding methods for producing compound(VI-a) or compound (VI-b) from a compound represented by the formula(V-a) (hereinafter referred to as compound (V-a)):

wherein R¹ has the same definition as the above; or from the lactoneform of compound (V-a) represented by the formula (V-b) (hereinafterreferred to as compound (V-b):

using a microorganism.

Specifically, Japanese Patent Application Laid-Open (kokai) No. 57-50894describes a method which uses filamentous fungi; both Japanese PatentApplication Laid-Open (kokai) No. 7-184670 and International PublicationWO96/40863 describe a method which uses Actinomycetes; and JapanesePatent No. 2672551 describes a method which uses recombinantActinomycetes. As is well known, however, since filamentous fungi andActinomycetes grow with filamentaous form by elongating hyphae, theviscosity of the culture in a fermentor increases.

This often causes a shortage of oxygen in the culture, and since theculture becomes heterogeneous, reaction efficiency tends to be reduced.In order to resolve this oxygen shortage and maintain homogenousness ofthe culture, the agitation rate of the fermentor should be raised, butby raising the agitation rate, hyphae are sheared and, as a result,activity of the microorganisms tends to decrease (Basic FermentationEngineering (Hakko Kogaku no Kiso) p. 169–190, P. E Stansbury, A.Whitaker, Japan Scientific Societies Press (1988)).

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a DNA encoding a novelhydroxylase, and an industrially advantageous method for producing acompound which inhibits HMG-CoA reductase and has an action of reducingthe level of serum cholesterol.

The present inventors considered that, if the hydroxylation of compound(I-a) or compound (I-b) could be carried out with a microorganismforming no hyphae, inconvenience such as the decrease of reactionefficiency due to the heterogeneity of the culture caused by hyphaeformation could be avoided, and that this would be industriallyadvantageous. Thus, as a result of intensive studies, the presentinventors have accomplished the present invention.

Thus, the present invention relates to the following (1) to (39).

Hereinafter, in the formulas, R¹ represents a hydrogen atom, asubstituted or unsubstituted alkyl, or an alkali metal, and R²represents a substituted or unsubstituted alkyl, or a substituted orunsubstituted aryl, unless otherwise specified.

(1) A protein which is derived from a microorganism belonging to thegenus Bacillus, and has an activity of producing compound (II-a) orcompound (II-b) from compound (I-a) or compound (I-b),

wherein the compound (I-a) is a compound represented by the formula(I-a):

the compound (I-b) is a lactone form of compound (I-a) and isrepresented by the formula (I-b):

the compound (II-a) is a compound represented by the formula (II-a):

the compound (I-b) is a lactone form of compound (II-a) and isrepresented by the formula (II-b):

(2) A protein which is derived from a microorganism belonging to thegenus Bacillus, and has an activity of producing compound (IV-a) orcompound (IV-b) from compound (III-a) or compound (II-b),wherein the compound (III-a) is a compound represented by the formula(III-a):

the compound (III-b) is a lactone form of compound (III-a) and isrepresented by the formula (III-b):

the compound (IV-a) is a compound represented by the formula (IV-a):

the compound (IV-b) is a lactone form of compound (IV-a) and isrepresented by the formula (IV-b):

(3) A protein which is derived from a microorganism belonging to thegenus Bacillus, and has an activity of producing compound (VI-a) orcompound (VI-b) from compound (V-a) or compound (V-b),wherein the compound (V-a) is a compound represented by the formula(V-a):

the compound (V-b) is a lactone form of compound (V-a) and isrepresented by the formula (V-b):

the compound (VI-a) is a compound represented by the formula (VI-a):

; andthe compound (VI-b) is a lactone form of compound (VI-a) and isrepresented by the formula (VI-b):

(4) A protein which is derived from a microorganism belonging to thegenus Bacillus, and has an activity of producing compound (VIII-a) orcompound (VIII-b) from compound (VII-a) or compound (VII-b),wherein the compound (VII-a) is a compound represented by the formula(VII-a):

the compound (VII-b) is a lactone form of compound (VII-a) and isrepresented by the formula (VII-b):

the compound (VIII-a) is a compound represented by the formula (VIII-a):

; andthe compound (VIII-b) is a lactone form of compound (VIII-a) and isrepresented by the formula (VIII-b):

(5) The protein according to any one of (1) to (4) above, wherein themicroorganism belonging to the genus Bacillus is a microorganismselected from B. subtilis, B. megaterium, B. laterosporus, B.sphaericus, B. pumilus, B. stearothermophilus, B. cereus, B. badius, B.brevis, B. alvei, B. circulars and B. macerans.(6) The protein according to any one of (1) to (5) above, wherein themicroorganism belonging to the genus Bacillus is a microorganismselected from B. subtilis ATCC6051, B. megaterium ATCC10778 B.megaterium ATCC11562, B. megaterium ATCC13402, B. megaterium ATCC15177,B. megaterium ATCC15450, B. megaterium ATCC19213, B. megaterium IAM1032,B. laterosporus ATCC4517, B. pumilus FERM BP-2064, B. badius ATCC14574,B. brevis NRRL B-8029, B. alvei ATCC6344, B. circulars NTCT-2610, and B.macerans NCIMB-9368.

(7) The protein according to any one of (1) to (5) above, wherein themicroorganism belonging to the genus Bacillus is a microorganismselected from Bacillus sp. FERM BP-6029 or Bacillus sp. FERM BP-6030.

(8) A protein having the amino acid sequence shown by SEQ ID NO: 1.

(9) A protein which has an amino acid sequence comprising deletion,substitution or addition of one or more amino acids in the amino acidsequence shown by SEQ ID NO: 1, and has an activity of producingcompound (II-a) or compound (II-b) from compound (I-a) or compound(I-b).(10) The protein according to (9) above, wherein the protein has theamino acid sequence shown by SEQ ID NO: 42 or 45.(11) The protein according to (9) above, wherein the compound (I-a) iscompound (III-a), the compound (I-b) is compound (III-b), the compound(II-a) is compound (IV-a), and the compound (II-b) is compound (IV-b).(12) The protein according to (9) above, wherein the compound (I-a) iscompound (V-a), the compound (I-b) is compound (V-b), the compound(II-a) is compound (VI-a), and the compound (II-b) is compound (VI-b).(13) The protein according to (9) above, wherein the compound (I-a) iscompound (VII-a), the compound (I-b) is compound (VII-b), the compound(II-a) is compound (VIII-a), and the compound (II-b) is compound(VIII-b).(14) An isolated DNA having the nucleotide sequence shown by SEQ ID NO:2.(15) An isolated DNA which hybridizes with the DNA according to (14)above under stringent conditions, and encodes a protein having anactivity of producing compound (II-a) or compound (I-b) from compound(I-a) or compound (I-b).(16) The DNA according to (15) above, wherein the DNA has a nucleotidesequence selected from the group consisting of the nucleotide sequencesshown by SEQ ID NOS: 41, 43 and 44.(17) An isolated DNA encoding the protein according to any one of (1) to(12) above.(18) The DNA according to (15) above, wherein the compound (I-a) iscompound (III-a), the compound (I-b) is compound (II-b), the compound(II-a) is compound (IV-a), and the compound (II-b) is compound (IV-b).(19) The DNA according to (15) above, wherein the compound (I-a) iscompound (V-a), the compound (I-b) is compound (V-b), the compound(II-a) is compound (VI-a), and the compound (II-b) is compound (VI-b).(20) The DNA according to (15) above, wherein the compound (I-a) iscompound (VII-a), the compound (I-b) is compound (VII-b), the compound(II-a) is compound (VIII-a), and the compound (II-b) is compound(VIII-b).(21) A recombinant DNA vector comprising the DNA according to any one of(14) to (20) above.(22) A transformant obtained by introducing the recombinant DNA vectoraccording to (21) above into a host cell.(23) The transformant according to (22) above, wherein the transformantbelongs to a microorganism selected from the genera Escherichia,Bacillus, Corynebacterium, and Streptomyces.(24) The transformant according to (22) or (23) above, wherein thetransformant belongs to microorganism selected from Escherichia coli,Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum,Corynebacterium ammoniagenes, Corynebacterium callunae and Streptomyceslividans.(25) A process for producing compound (II-a) or compound (II-b), whereinthe transformant according to any one of (22) to (24) above, a cultureof the transformant, or a treated product of the culture is used as anenzyme source, and the process comprises:allowing compound (I-a) or compound (I-b) to exist in an aqueous medium;allowing compound (II-a) or compound (II-b) to be produced andaccumulated in said aqueous medium; andcollecting compound (II-a) or compound (II-b) from said aqueous medium.(26) A process for producing compound (IV-a) or compound (IV-b), whereinthe transformant according to any one of (22) to (24) above, a cultureof the transformant, or a treated product of the culture is used as anenzyme source, and the process comprises:allowing compound (III-a) or compound (III-b) to exist in an aqueousmedium;allowing compound (IV-a) or compound (IV-b) to be produced andaccumulated in said aqueous medium; andcollecting compound (IV-a) or compound (IV-b) from said aqueous medium.(27) A process for producing compound (VI-a) or compound (VI-b), whereinthe transformant according to any one of (22) to (24) above, a cultureof the transformant, or a treated product of the culture is used as anenzyme source, and the process comprises:allowing compound (V-a) or compound (V-b) to exist in an aqueous medium;allowing compound (VI-a) or compound (VI-b) to be produced andaccumulated in said aqueous medium; andcollecting compound (VI-a) or compound (VI-b) from said aqueous medium.(28) A process for producing compound (VIII-a) or compound (VIII-b),wherein the transformant according to any one of (22) to (24) above, aculture of the transformant, or a treated product of the culture is usedas an enzyme source, and the process comprises:allowing compound (VII-a) or compound (VII-b) to exist in an aqueousmedium;allowing compound (VIII-a) or compound (VIII-b) to be produced andaccumulated in said aqueous medium; andcollecting compound (VIII-a) or compound (VIII-b) from said aqueousmedium.(29) The process according to (25) above, wherein the compound (II-b) isthe compound (II-b) obtained by forming a lacton from compound (II-a).(30) The process according to (25) above, wherein the compound (II-a) isthe compound (II-a) obtained by opening the lactone ring of compound(II-b).(31) The process according to (26) above, wherein the compound (IV-b) isthe compound (IV-b) obtained by forming a lacton from compound (IV-a).(32) The process according to (26) above, wherein the compound (IV-a) isthe compound (IV-a) obtained by opening the lactone ring of compound(IV-b).(33) The process according to (27) above, wherein the compound (VI-b) isthe compound (VI-b) obtained by forming a lacton from compound (VI-a).(34) The process according to (27) above, wherein the compound (VI-a) isthe compound (VI-a) obtained by opening the lactone ring of compound(VI-b).(35) The process according to (28) above, wherein the compound (VIII-b)is the compound (VIII-b) obtained by forming a lacton from compound(VIII-a).(36) The process according to (28) above, wherein the compound (VIII-a)is the compound (VIII-a) obtained by opening the lactone ring ofcompound (VIII-b).(37) The process according to any one of (25) to (28) above, wherein thetreated product of the culture of the transformant is a treated productselected from cultured cells; treated products such as dried cells,freeze-dried cells, cells treated with a surfactant, cells treated withan enzyme, cells treated by ultrasonication, cells treated by mechanicalmilling, cells treated by solvent; a protein fraction of a cell; and animmobilized products of cells or treated cells.(38) A process for producing a protein, which comprises culturing thetransformant according to any one of (22) to (24) above in a medium;producing and accumulating the protein according to any one of (1) to(12) above in the culture; and collecting said protein from saidculture.(39) An oligonucleotide corresponding to a sequence consisting of 5 to60 continuous nucleotides in a nucleotide sequence selected from thegroup consisting of the nucleotide sequences shown by SEQ ID NOS: 2, 41,43 and 44; or an oligonucleotide corresponding to a complementarysequence to said oligonucleotide.

The present invention will be described in detail below.

1. Obtaining of yjiB Gene

The DNA of the present invention can be obtained by PCR method [Science,230, 1350 (1985)] using the genome nucleotide sequence information of achromosome of Bacillus subtilis which has already been determined[http://www. pasteur.fr/Bio/SubtiList.html] and the information onBacillus subtilis yjiB gene deduced from said genome nucleotidesequence.

Specifically, the DNA of the present invention can be obtained by thefollowing method.

Bacillus subtilis (e.g., B. subtilis ATCC15563) is cultured by a usualmanner in a medium suitable for Bacillus subtilis, e.g. LB liquid medium[containing Bacto Trypton (produced by Difco) 10 g, yeast extract(produced by Difco) 5 g, and NaCl 5 g in 1 L of water; and adjusted topH 7.2]. After culturing, the cells are collected from the culture bycentrifugation.

A chromosomal DNA is isolated from the collected cells by a known method(e.g., Molecular Cloning 2^(nd) ed).

Using the nucleotide sequence information shown by SEQ ID NO:2, senseand antisense primers containing nucleotide sequences corresponding tothe DNA region encoding a protein of the present invention aresynthesized with a DNA synthesizer.

After amplification by PCR, in order to enable introduction of saidamplified DNA fragments into a plasmid, it is preferred that anappropriate restriction site such as BamHI, EcoRI or the like is addedat 5′ end of the sense and antisense primers.

Examples of combinations of said sense and antisense primers includecombination of DNAs having nucleotide sequences shown by SEQ ID NOS:13and 14.

Using chromosomal DNA as a template, PCR is performed with theseprimers, TaKaRa LA-PCR™ Kit Ver. 2 (TaKaRa), Expand™ High-Fidelity PCRSystem (Boehringer Mannheim) or the like by a DNA Thermal Cycler(Perkin-Elmer Japan).

When PCR is performed, for example, the following method can be carriedout. In the case where the above primer is a DNA fragment of 2 kb orless, each cycle consists of reaction steps of 30 seconds at 94° C., 30seconds to 1 minute at 55° C., and 2 minutes at 72° C. In the case wherethe above primer is a DNA fragment of more than 2 kb, each cycleconsists of reaction steps of 20 seconds at 98° C. and 3 minutes at 68°C. In any case, PCR is performed under conditions where the 30 cyclesare repeated, and then reaction is carried out for 7 minutes at 72° C.

The amplified DNA fragments are cut at the same restriction site as thesite which is formed using the above primers, and then the DNA fragmentsare fractioned and recovered by a method such as agarose gelelectrophoresis, sucrose density gradient ultracentrifugation and thelike.

Using the recovered DNA fragments, a cloning vector is produced by ausual method such as methods described in Molecular Cloning 2^(nd) ed.,Current Protocols in Molecular Biology, Supplement 1–38, John Wiley &Sons (1987–1997) (abbreviated as Current Protocols in Molecular Biology,Supplement hereinafter), DNA Cloning 1: Core Techniques, A PracticalApproach, Second Edition, Oxford University Press (1995), or by using acommercially available kit such as SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (produced by Life Technologies), ZAP-cDNASynthesis Kit (produced by Stratagene), etc., then the thus-producedcloning vector is used to transform Escherichia coli, e.g. E. coli DH5 αstrain (available from TOYOBO).

Examples of a cloning vector for the transformation of E. coli include aphage vector and plasmid vector insorfar as it is capable ofself-replicating in E. coli K12 strain. An expression vector for E. colican also be used as a cloning vector. Specifically, examples thereofinclude ZAP Express [produced by Stratagene, Strategies, 5, 58 (1992)],pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], LambdaZAP II (produced by Stratagene), λgt10, λgt11[DNA Cloning, A PracticalApproach, 1, 49 (1985)], λTriplEx (produced by Clonetech), λExCell(produced by Pharmacia), pT7T318U (produced by Pharmacia), pcD2 [H.Okayama and P. Berg; Mol. Cell. Biol., 3, 280 (1983)], pMW218 (producedby Wako Pure Chemical Industries), pUC118, pSTV28 (produced by Takara),pEG400 [J. Bac., 172, 2392 (1990)], pHMV1520 (produced by MoBiTec),pQE-30 (produced by QIAGEN), etc.

A plasmid containing a desired DNA can be obtained from the obtainedtransformed strain by usual methods described in e.g. Molecular Cloning2^(nd) edition, Current Protocols in Molecular Biology Supplement, DNACloning 1: Core Techniques, A Practical Approach, Second Edition, andOxford University Press (1995), etc.

Using the aforementioned method, a plasmid containing a DNA encoding aprotein which catalyzes reaction of producing compound (II-a) orcompound (I-b) from compound (I-a) or compound (I-b), can be obtained.

Examples of the plasmids include the below-mentioned pSyjiB.

Apart from the aforementioned method, a plasmid containing a DNAencoding a protein which catalyzes a reaction of producing compound(II-a) or compound (II-b) from compound (I-a) or compound (I-b) can beobtained also by a method wherein a chromosomal library of Bacillussubtilis is prepared with a suitable vector using E. coli as a host, andthe activity of producing compound (II-a) or compound (II-b) fromcompound (I-a) or compound (I-b) is measured on each strain of thislibrary.

The nucleotide sequence of the above-obtained gene can be used to obtainhomologues of the DNA from other prokaryotes or plants in the samemanner as mentioned above.

The DNA and DNA fragment of the present invention obtained in the abovemethod can be used to prepare oligonucleotides such as antisenseoligonucleotides, sense oligonucleotides etc. having a partial sequenceof the DNA of the present invention or such oligonucleotides containingRNAs. Alternatively, based on the sequence information of theabove-obtained DNA, these oligonucleotids can be synthesized with theabove DNA synthesizer.

Examples of the oligonucleotides include a DNA having the same sequenceas a contiguous 5 to 60 nucleotides in the nucleotide sequence of theabove DNA, or a DNA having a complementary sequence to said DNA. RNAshaving complementary sequences to these DNAs are also oligonucleotidesof the present invention.

Examples of said oligonucleotides include a DNA having the same sequenceas a contiguous 5 to 60 nucleotides sequence in the nucleotide sequencesshown by SEQ ID NOS:2, 41, 43 or 44, or a DNA having a complementarysequence to said DNA. If these are used as sense and antisense primers,the aforementioned oligonucleotides without extreme difference inmelting temperatures (Tm) and numbers of bases are preferably used.Specifically, examples thereof include oligonucleotides having anucleotide sequence shown by SEQ ID NOS: 3 to 39.

Furthermore, derivatives of these oligonucleotides (referred to asoligonucleotide derivative hereinafter) can also be used as the DNA ofthe present invention.

Oligonucleotide derivatives include a oligonucleotide derivative whosephosphate diester linkage is replaced by a phosphorothioate linkage, anoligonucleotide derivative whose phosphate diester linkage is replacedby a N3′-P5′ phosphoamidate linkage, an oligonucleotide derivative whoseribose and phosphate diester linkage is replaced by a peptide-nucleicacid linkage, an oligonucleotide derivative whose uracil is replaced byC-5 propinyl uracil, an oligonucleotide derivative whose uracil isreplaced by C-5 thiazol uracil, an oligonucleotide derivative whosecytosine is replaced by C-5 propinyl cytosine, an oligonucleotidederivative whose cytosine is replaced by phenoxazine-modified cytosine,an oligonucleotide derivative whose ribose is replaced by 2′-O-propylribose, or an oligonucleotide derivative whose ribose is replaced by2′-methoxy-ethoxyribose, etc. [Saibo Kogaku, 16, 1463 (1997).]

II. Method for Producing a Protein which Catalyzes a Reaction ofProducing Compound (II-a) or Compound (II-b) from Compound (I-a) orCompound (I-b)

In order to express the above-obtained DNA in a host cell, the desiredDNA fragment is cut into a fragment of suitable length containing saidgene using restriction enzymes or DNase enzymes, followed by insertingthe fragment into a site downstream of a promoter in an expressionvector, and then the expression vector is introduced into host cellssuitable for use of the expression vector.

The host cells may be any of bacteria, yeasts, animal cells, insectcells or the like insofar as they can express the objective gene.

As an expression vector, a vector capable of being autonomouslyreplicated in a host cell or capable of being integrated into achromosome, and containing a promoter at a site suitable fortranscription of the above objective gene, is used.

When prokaryotes such as bacteria are used as the host cell, theexpression vector for expressing the above DNA is preferably a vectorautonomously replicable in said cell and is a recombinant vectorcomposed of a promoter, a ribosome-binding sequence, the above DNA and atranscription termination sequence. A gene for regulating the promotermay be contained.

The expression vectors include pBTrp2, pBTac1, pBTac2 (all of which arecommercially available from Boehringer Mannheim), pKK233-2 (produced byPharmacia), pSE280 (produced by Invitrogen), pGEMEX-1 (produced byPromega), pQE-8 (produced by QIAGEN), pQE-30 (produced by QIAGEN),pKYP10 (Japanese Patent Application Laid-Open No. 58-110600), pKYP200[Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol.Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306(1985)], pBluescriptII SK(+), pBluescriptII SK(−) (produced byStratagene), pTrS30 (FERM BP-5407), pTrS32 (FERM BP-5408), pGEX(produced by Pharmacia), pET-3 (produced by Novagen), pTerm2 (U.S. Pat.No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735),pSupex, pUB110, pTP5, pC194, pUC18 [gene, 33, 103 (1985)], pUC19 [Gene,33, 103 (1985)], pSTV28 (produced by Takara), pSTV29 (produced byTakara), pUC118 (produced by Takara), pPA1 (Japanese Patent ApplicationLaid-Open No. 63-233798), pEG400 [J. Bacteriol., 172, 2392 (1990)],pQE-30 (produced by QIAGEN), PHY300 (produced by Takara), pHW1520(produced by MoBiTec), etc.

The promoter may be any one insofar as it can be expressed in a hostcell. Examples are promoters derived from E. coli, phage etc., such astrp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter andPSE promoter, and SP01 promoter, SP02 promoter, penP promoter and thelike. Artificially designed and modified promoters such as a Ptrp×2promoter having two Ptrp promoters in tandem, tac promoter, letIpromoter, and lacT7 promoter can also be used. Furthermore, xylApromoter for expression in Bacillus bacteria or P54-6 promoter forexpression in Corynebacterium bacteria can also be used.

Any ribosome binding sequences may be used insofar as they can work in ahost cell, and a plasmid in which the distance between a Shine-Dalgarnosequence and an initiation codon is adjusted to an appropriate distance(for example, 6 to 18 bases) may be preferably used.

For efficient transcription and translation, a protein which catalyzesthe reaction of producing compound (II-a) or compound (II-b) fromcompound (I-a) or compound (I-b) wherein the N-terminus or a partthereof is deleted may be fused to the N-terminus part of a proteinencoded by the expression vector, and the thus-obtained fused proteinmay be expressed. Such examples include the below-mentioned pWyjiB.

Although a transcription termination sequence is not necessarilyrequired for expression of the desired DNA, it is preferred to locatethe transcription termination sequence just downstream from thestructural gene.

Examples of prokaryotes include microorganisms belonging to the genusEscherichia, Corynebacterium, Brevibacterium, Bacillus, Microbacterium,Serratia, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Arthrobacter, Azotobacter, Chromatium, Erwinia,Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas,Rhodospirillum, Streptomyces, Synechococcus, and Zymomonas, preferablyEscherichia, Corynebacterium, Brevibacterium, Bacillus, Pseudomonas,Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter,Azotobacter, Chromatium, Erwinia, Methylobacterium, Phormidium,Rhodobacter, Rhodopseudomonas, Rhodospirillum, Streptomyces,Synechococcus, and Zymomonas.

Specific examples of the microorganisms include Escherichia coliXL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichiacoli DH5 α, Escherichia coli MC1000, Escherichia coli KY3276,Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101,Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49,Escherichia coli MP347, Escherichia coli NM522, Bacillus subtilisATCC33712, Bacillus megaterium Bacillus sp. FERM BP-6030, Bacillusamyloliquefacines, Brevibacterium ammmoniagenes, Brevibacteriumimmariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066,Brevibacterium flavum ATCC14067, Brevibacterium lactofermentumATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacteriumglutamicum ATCC14297, Corynebacterium acetoacidophilum ATCC13870,Corynebacterium callunae ATCC15991, Microbacterium ammoniaphilumATCC15354, Serratia ficaria, Serratia fonticola, Serratia liquefaciens,Serratia marcescens, Pseudomonas sp. D-0110, Agrobacterium radiobacter,Agrobacterium rhizogenes, Agrobacterium rubi, Anabaena cylindrical,Anabaena doliolum, Anabaena flos-aquae, Arthrobacter aurescens,Arthrobacter citreus, Arthrobacter globformis, Arthrobacterhydrocarboglutamicus, Arthrobacter mysorens, Arthrobacter nicotianae,Arthrobacter paraffineus, Arthrobacter protophormiae, Arthrobacterroseoparaffinus, Arthrobacter sulfurous, Arthrobacter ureafaciens,Chromatium buderi, Chromatium tepidum, Chromatium vinosum, Chromatiumwarmingii, Chromatium fluviatile, Erwinia uredovora, Erwinia carotovora,Erwinia ananas, Erwinia herbicola, Erwinia punctata, Erwinia terreus,Methylobacterium rhodesianum, Methylobacterium extorquens, Phormidiumsp. ATCC29409, Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodopseudomonas blastica, Rhodopseudomonas marina, Rhodopseudomonaspalustris, Rhodospirillum rubrum, Rhodospirillum salexigens,Rhodospirillum salinarum, Streptomyces ambofaciens, Streptomycesaureofaciens, Streptomyces aureus, Streptomyces fungicidicus,Streptomyces griseochromogenes, Streptomyces griseus, Streptomyceslividans, Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus, and Zymomonas mobilis.

The method for introducing the recombinant vector may be any method forintroducing DNA into the host cells described above. For examples,mention can be made of a method using calcium ions [Proc. Natl. Acad.Sci. USA, 69, 2110 (1972)], a protoplast method (Japanese PatentApplication Laid-Open No. 63-248394), an electroporation method, amethod described in Gene, 17, 107 (1982) and Molecular & GeneralGenetics, 168, 111 (1979), and the like.

If yeasts are used as the host cell, expression vectors such as YEp13(ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15 canbe exemplified.

Any promoter can be used insofar as they can be expressed in yeasts. Forexample, mention can be made of promoters such as PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter,heat shock protein promoter, MFα1 promoter, and CUP 1 promoter.

Examples of host cells include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans,and Schwanniomyces alluvius.

The method for introducing a recombinant vector may be any method forintroducing DNA into yeast, and examples include an electroporationmethod [Methods Enzymol., 194, 182 (1990)], a speroplast method [Proc.Natl. Acad. Sci. USA, 75, 1929 (1978)], a lithium acetate method [J.Bacteriol., 153, 163 (1983)] and a method describe in Proc. Natl. Acad.Sci. USA, 75, 1929 (1978).

If animal cells are used as the host cells, expression vectors such aspcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 (JapanesePatent Application Laid-Open No. 3-22979; Cytotechnology, 3, 133(1990)), pAS3-3 (Japanese Patent Application Laid-Open No. 2-227075),pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (Invitrogen), pREP4(Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], and pAGE210 canbe used.

The promoter to be used may be any promoter which can be expressed inanimal cells. Examples are a promoter for IE (immediate early) gene ofcytomegalovirus (human CMV), SV40 early promoter, retrovirus promoter,metallothionein promoter, heat shock promoter, Sr α promoter and thelike. Furthermore, an enhancer of the IE gene of human CMV may be usedtogether with a promoter.

Examples of animal cells include Namalwa cell, HBT5637 (Japanese PatentApplication Laid-Open No. 63-299), COS1 cell, COS7 cell, CHO cell andthe like.

The method for introducing a recombinant vector into animal cells may beany method for introducing DNA into animal cells. Examples of suchmethods include an electroporation method [Cytotechnology, 3, 133(1990)], a calcium phosphate method (Japanese Patent ApplicationLaid-Open No. 2-227075), a lipofection method [Proc. Natl. Acad. Sci.,USA, 84, 7413 (1987)], a method described in Virology, 52, 456 (1973),and the like. Obtaining and culturing of the transformant can beconducted according to methods described in Japanese Patent ApplicationLaid-Open No. 2-227075 or Japanese Patent Application Laid-Open No.2-257891.

If insect cells are used as the host cells, the protein can be expressedby methods described in Baculovirus Expression Vectors, A LaboratoryManual, Current Protocols in Molecular Biology Supplement 1–38(1987–1997); Bio/Technology, 6, 47 (1988) and the like.

That is, a recombinant gene transfer vector and a baculovirus areco-transfected into insect cells to obtain a recombinant virus in theculture supernatant of the insect cells, and then the insect cells areinfected with the recombinant virus whereby the protein can beexpressed.

Examples of the gene transfer vectors used in this method includepVL1392, pVL1393 and pBlueBacIII (all manufactured by Invitrogen).

As the baculovirus, it is possible to employ, e.g. Autographacalifornica nuclear polyhedrosis virus, that is, a virus infectinginsects of the family Barathra.

As the insect cells, it is possible to use Sf9, Sf21 [BaculovirusExpression Vectors, A Laboratory Manual, W.H. Freeman and Company, NewYork (1992)] which are oocytes of Spodopetera frugiperda and High 5(Invitrogen) which is oocyte of Trichoplusia ni, and the like.

As a method for co-transfering the aforesaid recombinant gene transfervector and the aforesaid baculovirus into insect cells for preparing therecombinant virus, for example, a calcium phosphate method (JapanesePatent Application Laid-Open No. 2-227075), a lipofection method [Proc.Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like may be used.

As a method for expressing gene, in addition to direct expression,secretory production, expression of a fusion protein and the like can becarried out according to the method described in Molecular Cloning2^(nd) edition.

When a protein has been expressed by yeasts, animal cells or insectcells, the protein to which a sugar or sugar chain is added can beobtained.

The thus-obtained transformant is cultured in a medium to produce andaccumulate proteins which catalyze the reaction of producing compound(II-a) or compound (II-b) from compound (I-a) or compound (I-b) in theculture, and the proteins are recovered from the culture, therebyproducing the protein which catalyzes production of compound (II-a) orcompound (II-b) from compound (I-a) or compound (I-b).

As a method for culturing in a medium the transformant for theproduction of the protein of the present invention which catalyzes thereaction of producing compound (II-a) or compound (II-b) from compound(I-a) or compound (I-b), conventional methods used for culturing atransformant in a host cell can be used.

If the transformant of the present invention is a prokaryote such as E.coli or an eukaryote such as yeast, the medium for culturing theseorganisms may be either a natural or synthetic medium insorfar as itcontains a carbon source, a nitrogen source, inorganic salts and thelike which can be assimilated by said organisms, and it allows efficientculturing of the transformant.

As a carbon source, any carbon source can be used as long as it can beassimilated by the microorganisms, including carbohydrates such asglucose, fructose, sucrose, or molasses containing those sources, starchor starch hydrolysates; organic acids such as acetic acid, propionicacid; and alcohols such as ethanol, propanol.

As a nitrogen source, the following can be used: ammonia; ammonium saltsof various inorganic acids and organic acids, such as ammonium chloride,ammonium sulfate, ammonium acetate, and ammonium phophate; othernitrogen-containing compounds; and peptone, meat extracts, yeastextracts, corn steep liquor, caselin hydrolysates, soy bean meal, soybean meal hydrolysates, various fermented cells and hydrolysatesthereof, and the like.

Examples of the inorganic substances include potassiumdihydrogenphosphate, potassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate and calcium carbonate.

The culturing is carried out under aerobic conditions by shake culturingor aeration-agitation culturing or the like. The culturing temperatureis preferably 15 to 50° C., and the culturing period is usually 16 hoursto 7 days. While culturing, pH is maintained at 3.0 to 9.0. The pHcontrol is conducted using an inorganic or organic acid, alkalinesolution, urea, calcium carbonate, ammonia and the like.

If necessary, antibiotics such as ampicillin and tetracycline may beadded to the medium while culturing.

When a microorganism transformed with an expression vector using aninductive promoter as a promoter is cultured, an inducer may beoptionally added to the medium. For example,isopropyl-β-D-thiogalactopyranoside (IPTG), indole acrylic acid (IAA) orxylose may be added to the medium respectively, when a microorganismtransformed with expression vectors containing lac promoter, trppromoter, or xylA promoter is used.

The medium for culturing the transformant obtained by using animal cellsas host cells may be a generally-used medium such as RPMI1640 medium[The Journal of the American Medical Association, 199, 519 (1967)],Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], 199 medium [Proceeding of the Society for the BiologicalMedicine, 73, 1 (1950)] or any one of these media further supplementedwith fetal calf serum.

Culturing is usually carried out for 1 to 7 days at pH 6 to 8 at 30 to40° C. in the presence of 5% CO₂.

If necessary, antibiotics such as kanamycin and penicillin may be addedto the medium while culturing.

The medium for culturing the transformant obtained by using insect cellsas host cells may be a generally-used medium such as TNM-FH medium(produced by Pharmingen), Sf-900 II SFM medium (produced by Gibco BRL),ExCell 400 and ExCell 405 [both are products of JRH Biosciences],Grace's Insect Medium [Grace, T. C. C., Nature, 195, 788 (1962)] or thelike.

Culturing is usually carried out at pH 6 to 7 at a temperature of 25 to30° C. for a period of 1 to 5 days.

If necessary, antibiotics such as gentamycin may be added to the mediumwhile culturing.

For isolating and purifying the protein which catalyzes a reaction ofproducing compound (II-a) or compound (II-b) from compound (I-a) orcompound (I-b) from the culture of the transformant of the presentinvention, any conventional methods for the isolation and purificationof enzymes can be performed.

For example, in the case where the protein of the present invention isexpressed in a soluble form in cells, after culturing, the cells arerecovered by centrifugation and suspended in an aqueous buffer, followedby disruption with ultrasonic disrupter, French Press, Manton-Gaulinhomogenizer, Dynomill or the like, thereby obtaining a cell-freeextract. From the supernatant obtained by centrifuging the cell-freeextract, a purified preparation can be obtained by using conventionalmethods for isolation and purification of enzymes alone or incombination, such as solvent extraction, salting-out or desalting withsulfate ammonium etc., precipitation with an organic solvent,anion-exchange chromatography on resin such as diethylaminoethyl(DEAE)-Sepharose, DIAION HPA-75 (produced by Mitsubishi ChemicalIndustries Ltd.) or the like, cation-exchange chromatography on resinsuch as S-Sepharose FF (Pharmacia) or the like, hydrophobicchromatography on resin such as butyl Sepharose, phenyl Sepharose or thelike, gel filtration using molecular sieve, affinity chromatography,chromatofocusing, and electrophoresis such as isoelectricelectrophoresis.

In the case where the protein is expressed in a form of an inclusionbody in cells, the cells are similarly recovered, disrupted andcentrifuged, thereby obtaining a precipitated fraction, and the proteinis recovered from the fraction in a usual manner. The recoveredinclusion body is solubilized with a protein denaturating agent. Thesolubilized solution is then diluted with or dialyzed against a solutionnot containing the protein denaturating agent or a solution containingthe protein denaturating agent at a concentration low enough not todenature the protein, whereby the protein is renatured to have normaltertiary structure, and its purified preparation can be obtained by thesame isolation and purification method as described above.

When the protein of the present invention or a saccharide modifiedderivatives thereof are extracellularly secreted, the protein or thederivatives to which saccharide chain is added, can be recovered fromthe supernatant of the culture. That is, the culture is subjected to anabove-mentioned process such as centrifugation and the like, therebyobtaining soluble fractions, then a purified preparation can be obtainedfrom said soluble fractions in the same manner as in the above.

Examples of the thus-obtained proteins include proteins having aminoacid sequences shown by SEQ ID NOS: 1, 42 or 45. Furthermore, theprotein expressed in the above manner can also be produced by chemicallysynthesis methods such as Fmoc method (fluorenyl methyloxycarbonylmethod) and tBoc method (t-butyloxycarbonyl method). Alternatively, theprotein can be obtained by synthesis using a peptide synthesizermanufactured by Sowa Trading (Advanced chemTech, USA), Perkin-ElmerJapan (Perkin Elmer, USA), Pharmacia Biotech (Pharmacia Biotech,Sweden), ALOKA (Protein Technology Instrument, USA), KURABO(Synthecell-Vega, USA), Japan PerSeptive Ltd. (PerSeptive, USA),Shimazu, etc.

III. Production of Compound (II-a) or Compound (I-b)

Using cells obtained by culturing the transformant obtained in above IIaccording to the method described in above II, a culture of said cells,a treated product of said culture, or an enzyme extracted from saidcells as enzyme sources, compound (II-a) or compound (II-b) can beproduced by allowing compound (I-a) or compound (I-b) to exist in anaqueous medium, allowing compound (II-a) or compound (I-b) to beproduced and accumulated in the above aqueous medium, and collectingcompound (II-a) or compound (I-b) from the above aqueous medium.

Examples of treated products of the culture of the cells include thetreated products of the cells such as dried cells, lyophiled cells,cells treated with surfactants, cells treated with enzymes, cellstreated with ultrasonication, cells treated with mechanical milling,cells treated with solvents; or protein fractions of the cells; orimmobilized products of said cell and said treated products of saidcells.

As a method for converting compound (I-a) or compound (I-b) intocompound (II-a) or compound (II-b), both of the following methods (a)and (b) can be used: (a) a method wherein the compound (I-a) or compound(I-b) is previously added to the medium for culturing cells; and (b) amethod wherein compound (I-a) or compound (I-b) is added to the mediumwhile culturing. Alternatively, a method wherein the enzyme sourceobtained from the cell culture is reacted with compound (I-a) orcompound (I-b) in the aqueous medium can be also used.

In a case where compound (I-a) or compound (I-b) is added to a medium inwhich a microorganism is to be cultured, 0.1 to 10 mg, preferably 0.2 to1 mg of compound (I-a) or compound (I-b) is added to 1 ml of medium atthe beginning of or at some midpoint of the culture. It is desired thatcompound (I-a) or compound (I-b) is added after it is dissolved in anorganic solvent such as methyl alcohol or ethyl alcohol.

In a case where a method of allowing an enzyme source obtained byculturing cells to act upon compound (I-a) or compound (I-b) in anaqueous medium, the amount of enzyme to be used depends on the specificactivity of the enzyme source or the like. For example, when a cultureof cells, cells, or a treated product thereof is used as an enzymesource, 5 to 1,000 mg, preferably 10 to 400 mg of enzyme source is addedper 1 mg of compound (I-a) or compound (I-b). The reaction is performedin an aqueous medium preferably at 20 to 50° C., and particularlypreferably at 25 to 37° C. The reaction period depends on the amount,specific activity and the like of an enzyme source to be used, and it isusually 2 to 150 hours, preferably 6 to 120 hours.

Examples of an aqueous medium include water, or buffers such asphosphate buffer, HEPES (N-2 hydroxyethylpiperazine-N-ethanesulfonate)buffer and Tris (tris(hydroxymethyl)aminomethane)hydrochloride buffer.An organic solvent may be added to the above buffers, unless it inhibitsreaction. Examples of organic solvent include acetone, ethyl acetate,dimethyl sulfoxide, xylene, methyl alcohol, ethyl alcohol and butanol. Amixture of an organic solvent and an aqueous medium is preferably used,for example when compound (I-b) is used.

In the case where compound (I-a) or compound (I-b) is added to theaqueous medium, compound (I-a) or compound (I-b) is dissolved in anaqueous medium capable of dissolving compound (I-a) or compound (I-b),and then is added to the medium. An organic solvent may be added to theabove buffers, unless it inhibits reaction. Examples of organic solventsinclude acetone, ethyl acetate, dimethyl sulfoxide, xylene, methylalcohol, ethyl alcohol and butanol.

Compound (I-b) and compound (II-b) can easily be converted into compound(I-a) and compound (II-a) respectively by a method for opening a lactonering as mentioned below. Likewise, compound (I-a) and compound (II-a)can easily be converted into compound (I-b) and compound (II-b)respectively by a method for producing lactone as mentioned below.

Examples of a method for opening a lactone ring include a method whichcomprises dissolving compound (I-b) or compound (II-b) in an aqueousmedium and adding thereto an acid or alkali. Examples of the aqueousmedium include water and an aqueous solution containing salts, whichdoes not inhibit the reaction, such as phosphate buffer, Tris buffer andthe like. The above aqueous solution may contain an organic solvent suchas methanol, ethanol, ethyl acetate and the like in a concentrationwhich does not inhibit the reaction. Examples of acid include aceticacid, hydrochloric acid and sulfuric acid, and examples of alkaliinclude sodium hydroxide, potassium hydroxide and ammonia.

Examples of a method for producing lactone include a method whichcomprises dissolving compound (I-a) or compound (II-a) in a non-aqueoussolvent and adding thereto an acid or base catalyst. As long as thenon-aqueous solvent is an organic solvent which does not substantiallycontain water and can dissolve compound (I-a) or compound (II-a), anytype of non-aqueous solvent can be used. Examples of non-aqueoussolvents include dichloromethane and ethyl acetate. As a catalyst, anycatalyst can be used, as long as it catalyzes lactonization and does notshow any actions other than lactonization on a substrate or a reactionproduct. Examples of the above catalyst include trifluoroacetic acid andpara-toluenesulfonic acid. Reaction temperature is not particularlylimited, but is preferably 0 to 100° C., and is more preferably 20 to80° C.

The collection of compound (II-a) or compound (II-b) from the reactionsolution can be carried out by any ordinary methods used in the field oforganic synthetic chemistry such as extraction with organic solvents,crystallization, thin-layer chromatography, high performance liquidchromatography, and the like.

As a method for detecting and quantifying compound (II-a) or compound(II-b) obtained by the present invention, any method can be used, aslong as the detection or quantification of compound (II-a) and/orcompound (II-b) can be performed. Examples thereof include ¹³C-NMRspectroscopy, ¹H-NMR spectroscopy, mass spectroscopy and highperformance liquid chromatography (HPLC).

In the present invention, some compounds of compound (I-a), compound(I-b), compound (II-a) and compound (II-b) can have stereoisomers suchas optical isomers. The present invention covers all possible isomersand mixtures thereof including these stereoisomers.

As compound (I-a), compound (III-a) is preferable, compound (V-a) ismore preferable, and compound (VII-a) is particularly preferable.

As compound (I-b), compound (II-b) is preferable, compound (V-b) is morepreferable, and compound (VII-b) is particularly preferable.

As compound (II-a), compound (IV-a) is preferable, compound (VI-a) ismore preferable, and compound (VIII-a) is particularly preferable.

As compound (II-b), compound (IV-b) is preferable, compound (VI-b) ismore preferable, and compound (VIII-b) is particularly preferable.

Alkyl is a linear or branched alkyl containing 1 to 10 carbon atoms,preferably 1 to 6 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl,hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, and various branched chain isomersthereof.

Examples of aryl include phenyl and naphtyl.

The substituent in the substituted alkyl may be 1 to 3 identical ordifferent groups, and examples thereof include halogens, hydroxy, amino,alkoxy and aryl.

The substituent in the substituted aryl may be 1 to 3 identical ordifferent groups, and examples thereof include halogens, hydroxy, amino,alkyl and alkoxy.

The alkyl moiety in alkoxy has the same definition as in the alkylmentioned above.

Alkali metal represents each element of lithium, sodium, potassium,rubidium, cesium or francium.

The examples of the present invention is described below, but thepresent invention is not limited to these examples.

BEST MODE FOR CARRYING-OUT OF THE INVENTION EXAMPLE 1 Obtaining of theDNA Encoding the Protein having an Activity of Producing Compound(VIII-a) or Compound (VIII-b) from Compound (VII-a) or Compound (VII-b)

100 mg of compound (VII-b) (produced by Sigma) was dissolved in 9.5 mlof methanol, and 0.5 ml of 1 mol/l sodium hydroxide was added. Themixture was stirred at room temperature for 1 hour. The obtainedreaction solution was dried to be solidified, and was dissolved byadding 5 ml of deionized water, followed by adjusting pH to about 7 withabout 0.1 ml of 1 mol/l hydrochloric acid. Then, 4.9 ml of deionizedwater was added to the mixture to obtain 10 ml of compound (VII-a),whose final concentration was 10 mg/ml (a compound wherein, in formula(VII-a), R¹ is sodium).

Bacillus subtilis Marburg168 strain (ATCC15563) was inoculated with 1platinum loop in a 10 ml LB liquid medium, and cultured at 30° C.overnight. After culturing, cells were collected from the obtainedculture solution by centrifugation.

A chromosomal DNA was isolated and purified from the cells in a usualmanner.

Sense and antisense primers having a combination of nucleotidesequences: SEQ ID NOS: 3 and 4, SEQ ID NOS: 5 and 6, SEQ ID NOS: 7 and8, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14,and SEQ ID NOS: 15 and 16, were synthesized with a DNA synthesizer.

Using the chromosomal DNA as a template, PCR was performed with theseprimers and with TaKaRa LA-PCR™ Kit Ver.2 (produced by TAKARA), Expand™High-Fidelity PCR System (produced by Boehringer Mannheim) or Taq DNApolymerase (produced by Boelinnger) using a DNA Thermal Cycler (producedby Perkin-Elmer Japan).

PCR was performed for 30 cycles in which each cycle consists of reactionsteps of 30 seconds at 94° C., 30 seconds at 55° C. and 2 minutes at 72°C. for DNA fragments of 2 kb or less; and 20 seconds at 98° C., 3minutes at 68° C. for DNA fragments of more than 2 kb, and then reactionwas carried out for 7 min at 72° C.

Among DNA fragments amplified by PCR, the DNA fragment (containing biolgene) amplified by a combination of primers of SEQ ID NOS:3 and 4 wasdigested with restriction enzymes EcoRI and SalI, DNA fragment(containing cypA gene) amplified by a combination of primers of SEQ IDNOS:5 and 6 was digested with XbaI and SmaI, DNA fragment (containingcypX gene) amplified by a combination of primers of SEQ ID NOS:7 and 8was digested with SmaI and SalI, DNA fragment (containing pksS gene)amplified by a combination of primers of SEQ ID NOS:9 and 10 wasdigested with EcoRI and SalI, DNA fragment (containing yet0 gene)amplified by a combination of primers of SEQ ID NOS:11 and 12 wasdigested with XbaI and BglII, DNA fragment (containing yjiB gene)amplified by a combination of primers of SEQ ID NOS:13 and 14 wasdigested with XbaI and SmaI, and DNA fragment (containing yrhJ gene)amplified by a combination of primers of SEQ ID NOS:15 and 16 wasdigested with XbaI and SmaI, respectively.

After digestion, the DNA fragments treated with the restriction enzymeswere subjected to agarose gel electrophoresis to obtain the DNAfragments treated with various restriction enzymes.

A vector plasmid pUC119 (produced by TAKARA) was digested withrestriction enzymes SalI and EcoRI, then subjected to agarose gelelectrophoresis to obtain a SalI-EcoRI treated pUC119 fragment.Similarly, a vector plasmid pUC119 was digested with restriction enzymesSalI and SmaI, then subjected to agarose gel electrophoresis to obtain aSalI-SmaI treated pUC119 fragment.

pSTV28 (produced by TAKARA) was digested with restriction enzymes XbaIand SmaI, then subjected to agarose gel electrophoresis to obtain aXbaI-SmaI treated pSTV28 fragment. Similarly, a vector plasmid pSTV28was digested with restriction enzymes XbaI and BamHI, then subjected toagarose gel electrophoresis to obtain a XbaI-BamHI treated pSTV28fragment.

The thus-obtained EcoRI-SalI treated DNA fragment (amplified by PCR witha combination of primers of SEQ ID NOS:3 and 4) was mixed with theSalI-EcoRI treated pUC119 fragment, XbaI-SmaI treated DNA fragment(amplified by PCR with a combination of primers of SEQ ID NOS:5 and 6)was mixed with the XbaI-SmaI treated pSTV28 fragment, SmaI-SalI treatedDNA fragment (amplified by PCR with a combination of primers of SEQ IDNOS:7 and 8) was mixed with the SalI-SmaI treated pUC119 fragment,EcoRI-SalI treated DNA fragment (amplified by PCR with a combination ofprimers of SEQ ID NOS:9 and 10) was mixed with the SalI-EcoRI treatedpUC119 fragment, XbaI-BglII treated DNA fragment (amplified by PCR witha combination of primers of SEQ ID NOS:11 and 12) was mixed with theXbaI-BamHI treated pSTV28 fragment, XbaI-SmaI treated DNA fragment(amplified by PCR with a combination of primers of SEQ ID NOS:13 and 14)was mixed with the XbaI-SmaI treated pSTV28 fragment, XbaI-SmaI treatedDNA fragment (amplified by PCR with a combination of primers of SEQ IDNOS:15 and 16) was mixed with XbaI-SmaI treated pSTV28 fragment,respectively. After ethanol precipitation, the obtained DNA precipitateswere dissolved in 5 μl of distilled water, and a ligation reaction wascarried out to obtain each recombinant DNA.

Using the recombinant DNA, E. coli (purchased from TOYOBO) DH5 α strainis transformed by a usual method, then the transformant was plated to aLB agar medium [containing Bacto Trypton (produced by Difco) 10 g,Bactoyeast extract (produced by Difco) 5 g, NaCl 5 g in 1 L; andadjusted to pH7.4 with 1 mol/l NaOH such that the agar is adjusted to1.5%] containing 100 μg/ml ampicillin in the case where the pUC119 isused as a vector plasmid; and to a LB agar medium containing 25 μg/mlchloramphenicol in the case where the pSTV28 is used as a vectorplasmid, followed by culturing for 2 days at 25° C.

Several colonies of the grown ampicillin-resistant orchloramphenicol-resistant transformants were selected, inoculated in 10ml LB liquid medium [which contains Bacto Trypton (produced by Difco) 10g, Bactoyeast extract (produced by Difco) 10 g and NaCl 5 g in 1 L; andis adjusted to pH 7.4 with 1 mol/l NaOH], and then cultured whileshaking for 2 days at 25° C.

The obtained culture was centrifuged to recover cells.

A plasmid was isolated from the cells in a usual manner.

The structure of the isolated plasmid was examined by cleaving it withvarious restriction enzymes and the nucleotide sequences weredetermined, thereby confirming that the desired DNA fragment wasinserted in the plasmid. The plasmids obtained by linking the DNAfragment (amplified by PCR with a combination of primers of SEQ ID NOS:3and 4) treated with EcoRI-SalI to pUC119 fragment treated withSalI-EcoRI, was named pUbiol, the plasmids obtained by linking DNAfragment (amplified by PCR with a combination of primers of SEQ ID NOS:5and 6) treated with XbaI-SmaI to pSTV28 fragment treated with XbaI-SmaIwas named pScypA, the plasmids obtained by linking DNA fragment(amplified by PCR with a combination of primers of SEQ ID NOS:7 and 8)treated with SmaI-SalI to pUC119 fragment treated with SalI-SmaI wasnamed pUcypX, the plasmids obtained by linking DNA fragment (amplifiedby PCR with a combination of primers of SEQ ID NOS:9 and 10) treatedwith EcoRI-SalI to pUC119 fragment treated with SalI-EcoRI was namedpUpksS, the plasmids obtained by linking DNA fragment (amplified by PCRwith a combination of primers of SEQ ID NOS:11 and 12) treated withXbaI-BglII to pSTV28 fragment treated with XbaI-BamHI was named pSyetO,the plasmids obtained by linking DNA fragment (amplified by PCR with acombination of primers of SEQ ID NOS:13 and 14) treated with XbaI-SmaIto pSTV28 fragment treated with XbaI-SmaI was named pSyjiB, the plasmidsobtained by linking DNA fragment (amplified by PCR with a combination ofprimers of SEQ ID NOS:15 and 16) treated with XbaI-SmaI to pSTV28fragment treated with XbaI-SmaI was named pSyrhJ, respectively.

E. coli DH5 α containing the thus-obtained plasmid, E. coli DH5 αcontaining pUC119 or pSTV28, and E. coli DH5 α containing no plasmidwere inoculated respectively in 3 ml of LB liquid medium (to which adrug which corresponds to a drug-resistant gene in a vector plasmid wasadded) and cultured while shaking for 12 hours at 28° C. The culturesolution (0.5 ml) was inoculated to a LB liquid medium (to which a drugwhich corresponds to a drug-resistant gene was added) containing 1%glucose and 1% CaCO₃, and was cultured while shaking for 12 hours at 28°C. The culture solution (1 ml) was poured into an assist tube (producedby ASSIST), then glucose and the previously obtained compound (VII-a)(wherein R¹ is a Na) were added to a final concentration of 1% and 100mg/l, respectively, followed by shaking for 24 hours at 28° C. Uponcompletion of the reaction, cells were removed by centrifugation, thenthe obtained supernatant was thoroughly shaken with addition of the sameamount of ethyl acetate. The upper ethyl acetate layer was separatedfrom the solution by centrifugation, then the ethyl acetate layer wasevaporated to dryness by a centrifugal evaporator. The dried matter wasdissolved in one-fifths volume of methanol relative to that of the firstculture supernatant, and subjected to a HPLC analysis [column; InertsilODS-2 (5 μm, 4×250 mm, manufactured by GL science), column temperature;60° C., mobile phase; acetonitrile: water: phosphoric acid=55:45:0.05,flow rate: 0.9 ml/min, detection wavelength: 237 nm] to detect andquantify the compound (VIII-a) (wherein R¹ is Na). The results are shownin Table 1.

TABLE 1 Plasmid Compound (VIII-a) (mg/l) None 0 pUC119 0 pSTV28 0 pUbioI0 pScypA 0 pUcypX 0 pUpksS 0 pSyetO 0 pSyjiB 0.6 pSyrhJ 0

EXAMPLE 2 Expression of yjiB Gene in Bacillus subtilis as a Host Celland Confirmation of Activity of the Protein Encoded by Said Gene

Sense and antisense primers having a combination of nucleotide sequencesshown by SEQ ID NOS:17 and 18, SEQ ID NOS:19 and 20, SEQ ID NOS:21 and22, SEQ ID NOS:23 and 24, SEQ ID NOS:25 and 26, SEQ ID NOS:27 and 28,and SEQ ID NOS:29 and 30, were synthesized with a DNA synthesizer.

Using the chromosomal DNA of Bacillus subtilis obtained in Example 1 asa template, PCR was performed with these primers and with TaKaRa LA-PCR™Kit Ver.2 (produced by TAKARA), Expand™ High-FidelityPCR System(produced by Boehringer Mannheim) or Taq DNA polymerase (produced byBoellinnger) using a DNA Thermal Cycler (produced by Perkin-ElmerJapan).

PCR was performed for 30 cycles under the conditions where one cycleconsists of the reaction steps of 30 seconds at 94° C., 30 seconds at55° C. and 2 minutes at 72° C. for the DNA fragments of 2 kb or less,and 20 seconds at 98° C. and 3 minutes at 68° C. for the DNA fragmentsof more than 2 kb, and then a reaction was carried out for 7 minutes at72° C.

Among DNA fragments amplified by PCR, the DNA fragment (containing biolgene) amplified by a combination of primers of SEQ ID NOS:17 and 18 wasdigested with rectriction enzymes SpeI and BamHI, DNA fragment(containing cypA gene) amplified by a combination of primers of SEQ IDNOS:19 and 20 was digested with SpeI and BamHI, DNA fragment (containingcypX gene) amplified by a combination of primers of SEQ ID NOS:21 and 22was digested with SpeI and NruI, DNA fragment (containing pksS gene)amplified by a combination of primers of SEQ ID NOS:23 and 24 wasdigested with SpeI and BamHI, DNA fragment (containing yet0 gene)amplified by a combination of primers of SEQ ID NOS:25 and 26 wasdigested with SpeI and BamHI, DNA fragment (containing yjiB gene)amplified by a combination of primers of SEQ ID NOS:27 and 28 wasdigested with SpeI and BamHI, DNA fragment (containing yrhJ gene)amplified by a combination of primers of SEQ ID NOS:29 and 30 wasdigested with SpeI and BamHI, respectively.

After digestion, the DNA fragments treated with the restriction enzymeswere subjected to agarose gel electrophoresis to obtain the DNAfragments treated with each restriction enzyme.

A vector plasmid pWH1520 (produced by MoBiTec) was digested withrestriction enzymes SpeI and BamHI, then subjected to agarose gelelectrophoresis to obtain a SpeI-BamHI treated pWH1520 fragment.Similarly, a vector plasmid pWH1520 was digested with restrictionenzymes SpeI and NruI, then subjected to agarose gel electrophoresis toobtain a SpeI-NruI pWH1520 fragment.

The thus-obtained SpeI-BamHI treated DNA fragments (amplified by PCRwith a combination of primers of SEQ ID NOS:17 and 18, SEQ ID NOS:19 and20, SEQ ID NOS:23 and 24, SEQ ID NOS:25 and 26, SEQ ID NOS:27 and 28,and SEQ ID NOS:29 and 30) were mixed with the SpeI-BamHI treated pWF1520fragment; SpeI-NruI treated DNA fragment (amplified by PCR with acombination of primers of SEQ ID NOS:21 and 22) was mixed with SpeI-NruIpWF1520 fragment, respectively. After ethanol precipitation, theobtained DNA precipitates were dissolved in 5 μl of distilled water, anda ligation reaction was carried out to obtain each recombinant DNA.

Using the recombinant DNA, E. coli (purchased from TOYOBO) DH5 α strainwas transformed by a usual method, then plated to a LB agar mediumcontaining 10 μg/ml of tetracycline, and cultured for 2 days at 25° C.Cells were recovered from the obtained culture by centrifugation.

A plasmid was isolated from the cells in a usual manner.

The structure of the isolated plasmid was examined by cleaving it withvarious restriction enzymes and the nucleotide sequences thereof weredetermined, thereby confirming that the desired DNA fragment wasinserted in the plasmid. The plasmid obtained by linking the DNAfragment amplified by PCR with a combination of primers of SEQ ID NOS:17and 18 to pWH1520 was named as pWbioI; the plasmid obtained by linkingthe DNA fragment amplified by PCR with a combination of primers of SEQID NOS:19 and 20 to pWH1520 was named as pWcypA; the plasmid obtained bylinking the DNA fragment amplified by PCR with a combination of primersof SEQ ID NOS:21 and 22 to pWH1520 was named as pWcypX; the plasmidobtained by linking the DNA fragment amplified by PCR with a combinationof primers of SEQ ID NOS:23 and 24 to pWH1520 was named as pWpksS; theplasmid obtained by linking the DNA fragment amplified by PCR with acombination of primers of SEQ ID NOS:25 and 26 to pWH1520 was named aspWyet0; the plasmid obtained by linking the DNA fragment amplified byPCR with a combination of primers of SEQ ID NOS:27 and 28 to pWH1520 wasnamed as pWyjiB; the plasmid obtained by linking the DNA fragmentamplified by PCR with a combination of primers of SEQ ID NOS:29 and 30to pWH1520 was named as pWyrhJ, respectively.

The thus-obtained plasmids and the vector plasmid pWH1520 wereintroduced in a Bacillus subtilis ATCC33712 strain according to themethod by S. chang and S. N. cohen [S. chang and S. N. cohen: Mol. Gen.Genet., 168, 111 (1979).]

That is, ATCC33712 strain was inoculated in a thick tube containing 5 mlof Pen medium (where 1.75 g of Difco Antibiotic medium No. 3 wasdissolved in 100 ml of water and sterilized in an autoclave), thencultured with shaking at 37° C. overnight. Total cells culturedovernight in 300 ml Erlenmeyer flask containing 100 ml of Pen mediumwere then inoculated and cultured with shaking for 3 hours at 37° C. tobe grown until reaching a metaphase of exponential growth. The culturewas centrifuged for 10 minutes at 5000 rpm in germ-free conditions toprecipitate the cells. After removing the supernatant, the cells weresuspended in 4.5 ml of SMMP [mixture comprising equal amount of 2×SMMP(where sucrose 34.2 g, maleic acid 0.464 g, magnesium chloride.6H₂O0.813 g were dissolved in water, which was adjusted to pH6.5 with sodiumhydroxide, then the final volume of 100 ml was sterilized in anautoclave) and 4×Pen medium (where 7 g of Difco Antibiotic medium No. 3was dissolved in 100 ml of water and sterilized in an autoclave)],followed by addition of 0.5 ml of lysozyme solution [where 10 mg oflysozyme (produced by EIKAGAKU corp.) was dissolved in 0.5 ml of SMMP,and sterilized with a millipore filter having a pore size of 0.45 μm],and the mixture was slowly shaken for 2 hours at 37° C. Aftermicroscopically confirming that not less than 90% cells becameprotoplast, the protoplasts were centrifuged for 20 minutes at 3000 rpmto be precipitated. The supernatant was removed, and the obtainedprotoplasts were resuspended in 5 ml of SMMP. The protoplasts werecollected by centrifugation for 20 minutes at 3000 rpm, and suspended in2 ml of SMMP to prepare a protoplast suspension of a recipient strainfor transformation.

Approximately 1 μg of plasmid DNA was dissolved in SMMP, and thoroughlymixed with 0.5 ml of protoplast suspension. Immediately after mixing,1.5 ml of 40% polyethylene glycol solution [where 40 g of polyethyleneglycol 6000 (Nacalai tesque) was dissolved in 2×SMMP, and water wasadded thereto to become the volume of 100 ml, followed by sterilizationin an autoclave] was added thereto and thoroughly mixed. After standingat room temperature for 2 minutes, 5 ml of SMMP was added and mixed, andthe mixture was centrifuged for 20 minutes at 3000 rpm. After removingthe supernatant, the precipitated protoplasts were suspended in 1 ml ofSMMP, then slowly shaken for 3 hours at 30° C. After dilution with SMMPas appropriate, the protoplasts were applied to a DM3 medium [in which45 ml of 80 g/L bactoagar (produced by Difco), 50 ml of 50 g/L casaminoacid, 250 ml of 338 g/L sodium succinate .6H₂O (pH7.3), 50 ml phosphatebuffer (35 g/L potassium hydrogen phosphate, 15 g/L potassium dihydrogenphosphate), 25 ml of 100 g/L yeast extract, 10 ml of 203 g/L magnesiumchloride.6H₂O, 25 ml of 100 g/L glucose were respectively sterilized inan autoclave and mixed, then 3.5 ml of 20 mg/ml bovine serum albuminsterilized with millipore filter having a pore size of 0.45 μm was addedthereto] wherein the medium containing drugs (in case of tetracycline,it was added to the final volume of 10 μg/ml). The protoplasts werecultured for 1 to 2 days at 37° C. to obtain the transfected strain.

Thus, B. subtilis ATCC33712 strains having each of the above plasmidswere obtained.

The obtained transformants and ATCC33712 strain having no plasmid wereinoculated respectively in 3 ml LB liquid media (wherein 10 mg/ltetracycline was added to a plasmid-containing strain), and culturedwith shaking for 24 hours at 30° C. 0.25 ml of this culture solution wasinoculated in a test tube containing a 5 ml of TB medium [Bacto Trypton(produced by Difco) 1.4%, Bacto yeast extract (produced by Difco) 2.4%,KH₂PO₄O 0.231%, and K₂HPO₄ 1.251%, adjusted to pH7.4 with 1 mol/l sodiumhydroxide], and cultured with shaking for 3 hours at 30° C. After 3hours, 1 ml of the culture was transferred to an assist tube No. 60.540S(produced by ASSIST) and 40 μl of 50% sterilized xylose solution wasadded thereto, followed by culturing with shaking for 3 hours. Then, thecompound (VII-a) (wherein R is Na) obtained in Example 1 was added toeach test tube to the final concentration of 0.2 mg/ml, and the mixturewas cultured whith shaking for 16 hours at 30° C.

Upon completion of reaction, the reaction solution was adjusted to pH3.5 with acetic acid. 1 ml of ethyl acetate was added to 0.5 ml of thisreaction solution, and the mixture was shaken for 1 hour. After shaking,the reaction solution was centrifuged for 5 minutes at 3000 rpm to beseparated into 2 layers, and the upper ethyl acetate layer wasrecovered, the solvent was removed by a centrifugal evaporator, and theresidue was dissolved in 0.5 ml of methanol.

Using an aliquot of this methanol solution, HPLC analysis was performedas in Example 1 to detect and quantify compound (VIII-a) (wherein R¹ isNa). The results are shown in Table 2.

TABLE 2 Plasmid Compound (VIII-a) (mg/l) None 0.5 pWH1520 0.5 pWbioI 0.5pWcypA 0.5 pWcypX 0.5 pWpksS 0.5 pWyetO 0.5 pWyjiB 24.6 pWyrhJ 0.5

As seen from the results of Example 1 and 2, it is obvious that activityof producing compound (VIII-a) or compound (VIII-b) from compound(VII-a) or compound (VII-b) is encoded by yjiB gene.

The DNA fragment amplified by PCR with a combination of primers of SEQID NO:27 and 28 above, contained the nucleotide sequence shown by SEQ IDNO:2; and said nucleotide sequence contained a nucleotide sequenceencoding a protein having the amino acid sequence shown by SEQ ID NO:1.

EXAMPLE 3 Expression of yjiB Gene Using Bacillus megaterium as a HostCell and Production of Compound (VIII-a)

pWyjiB prepared in Example 2 was introduced into Bacillus megaterium(produced by MoBiTec) and Bacillus sp. FERM BP-6030 in the same manneras is described for transformation of Bacillus subtilis in Example 2.

The obtained transformant and a host cell having no plasmid werecultured and reaction was carried out in the same manner as in Example2, and the amount of produced compound (VIII-a) was measured. Theresults are shown in Table 3.

TABLE 3 Host Plasmid Compound (VIII-a) (mg/l) B. megaterium none 2.0 (asabove) pWyjiB 27.2 FERM BP-6030 none 4.5 (as above) pWyjiB 30.3

EXAMPLE 4 Preparation of the Plasmid for Expressing the Protein whichProduces Compound (VIII-a) in Coryne-Form Bacteria

To allow efficient expression of yjiB gene obtained in Example 1 incoryne-form bacteria, DNAs having nucleotide sequences shown by SEQ IDNOS:31, 32, 33, 34, 35, 36, 37, 38 and 39 were synthesized with a DNAsynthesizer.

The plasmid pR1109 DNA in which the DNA fragment comprising a promotersequence p54-6 (GenBank AJ132582) for expression in coryne-form bacteriaand having the nucleotide sequence shown by SEQ ID NO:40 was insertedinto a Sse83871-BamHI site of a plasmid vector pCS299P (Japanese PatentApplication No. 11-110437), was prepared in a usual manner from E. coliNM522 strain transformed with this plasmid.

Using pWyjiB DNA obtained in Example 2 as a template, PCR was performedwith DNA primers having nucleotide sequences shown by SEQ ID NOS:31 and32 and with Taq DNA polymerase (produced by TAKARA) using a DNA ThermalCycler 480 (produced by Perkin-Elmer Japan).

PCR was performed for 25 cycles in which each cycle consists of reactionsteps of 30 seconds at 96° C., 45 seconds at 50° C. and 3 minutes at 72°C.

DNA fragment amplified by PCR was digested with SalI and BamHI andsubjected to agarose gel electrophoresis, and an approximately 1.2 kbDNA fragment was purified in a usual manner to obtain a SalI-BamHItreated DNA fragment.

The above-obtained plasmid pRI109 DNA was digested with restrictionenzymes SalI and BamHI and subjected to agarose gel electrophoresis, andan approximately 6 kb DNA fragment was purified in a usual manner toobtain a SalI-BamHI treated pRI109 fragment.

The above-obtained SalI-BamHI treated DNA fragment and SalI-BamHItreated pRI109 fragment were mixed, and ligation reaction was carriedout to obtain the recombinant DNA.

Using the recombinant DNA, E. coli DH5 α(purchased from TOYOBO) wastransformed by a usual method, then plated to a LB agar mediumcontaining 20 μg/ml kanamycin and cultured for 1 day at 30° C. to obtainthe transformant.

A plasmid was isolated from the transformant in a usual manner. Usingthe isolated plasmid DNA as a template, and using DNAs having nucleotidesequences shown by SEQ ID NOS:33, 34, 35, 36 and 37 as primersrespectively, the nucleotide sequences of the inserted DNA fragmentswere determined with a DyeTerminator Cycle Sequencing Kit (produced byApplied Biosystem) and 373A sequencer (produced by Applied Biosystem),then the plasmid in which the nucleotide sequence shown by SEQ ID NO:41was inserted between SalI and BamHI sites of pRI109 was named pRIyjiB.

The nucleotide sequence shown by SEQ ID NO:41 contained the nucleotidesequence which encoded the protein having the amino acid sequence shownby SEQ ID NO:42.

Using the chromosomal DNA of Bacillus subtilis Marburg168 strain(ATCC15563) obtained in Example 1 as a template, PCR was performed withDNA primers having nucleotide sequences shown by SEQ ID NOS:38 and 39,and with LA-Taq DNA polymerase (produced by TAKARA) using a DNA ThermalCycler 480 (produced by Perkin-Elmer Japan).

PCR was performed for 30 cycles in which each cycle consists of reactionsteps of 30 seconds at 96° C., 30 seconds at 55° C. and 2 minutes at 72°C., and then a reaction was carried out for 7 minutes at 72° C.

The DNA fragment amplified by PCR was mixed with pT7Blue (produced byTAKARA), and ligation reaction was carried out to obtain the recombinantDNA.

Using the recombinant DNA, E. coli DH5 α (purchased from TOYOBO) wastransformed by a usual method, then plated to a LB agar mediumcontaining 100 μg/ml ampicillin and cultured for 1 day at 30° C. toobtain the transformant.

A plasmid was isolated from the transformant by a usual method. Thestructure of the isolated plasmid was examined by cleaving it withvarious restriction enzymes, thereby confirming that the desired DNAfragment was inserted in the plasmid, and the plasmid was named aspTSYN2-72.

The pTSYN2-72 DNA was digested with XhoI and BamHI and subjected toagarose gel electrophoresis, and then an approximately 1.2 kb DNAfragment was purified by a usual method to obtain a XhoI-BamHI treatedDNA fragment.

The plasmid pRI109 DNA was digested with restriction enzymes SalI andBamHI and subjected to agarose gel electrophoresis, and then anapproximately 6 kb DNA fragment was purified by a usual method to obtaina SalI-BamHI treated pRI109 fragment.

The above-obtained XhoI-BamHI treated DNA fragment and SalI-BamHItreated pRI109 fragment were mixed, and the ligation reaction wascarried out to obtain the recombinant DNA.

Using the recombinant DNA, E. coli DH5 α (purchased from TOYOBO) wastransformed by a usual method, then plated to a LB agar mediumcontaining 20 μg/ml kanamycin and cultured for 1 day at 30° C. to obtaina transformant.

A plasmid was isolated from the transformant by a usual method. Usingthe isolated plasmid DNA as a template, and using DNAs having nucleotidesequences shown by SEQ ID NOS:33, 34, 35, 36 and 37, the nucleotidesequences of the inserted DNA fragment were determined with aDyeTerminator Cycle Sequencing Kit (produced by Applied Biosystem) and373A sequencer (produced by Applied Biosystem), and the plasmid in whichthe nucleotide sequence shown by SEQ ID NO:43 was inserted betweenSalI-BamHI site of pRI109, was named pSYN2-72.

The nucleotide sequence shown by SEQ ID NO:43 contained the nucleotidesequence which encodes the protein having the amino acid sequence shownby SEQ ID NO:1.

Using pWyjiB DNA obtained in Example 2 as a template, PCR was performedwith DNA primers having nucleotide sequences shown by SEQ ID NO:38 and39, and with Z-Taq DNA polymerase (produced by TAKARA) using a DNAThermal Cycler 480 (produced by Perkin-Elmer Japan).

PCR was performed for 25 cycles in which each cycle consists of reactionsteps of 20 seconds at 98° C., 20 seconds at 55° C. and 30 minutes at72° C.

The DNA fragment amplified by PCR was digested with XhoI and BamHI andsubjected to agarose gel electrophoresis, and then an approximately 1.2kb DNA fragment was purified by a usual method to obtain a XhoI-BamHItreated DNA fragment.

The plasmid pRI109 DNA was digested with restriction enzymes SalI andBamHI and subjected to agarose gel electrophoresis, then anapproximately 6 kb DNA fragment was purified by a usual method to obtaina SalI-BamHI treated pRI109 fragment.

The above-obtained XhoI-BamHI treated DNA fragment and SalI-BamHItreated pRI109 fragment were mixed, and ligation reaction was carriedout to obtain the recombinant DNA.

Using the recombinant DNA, E. coli DH5 α (purchased from TOYOBO) wastransformed by a usual method, then plated to a LB agar mediumcontaining 20 μg/ml kanamycin and cultured for 1 day at 30° C. to obtainthe transformant.

A plasmid was isolated from the transformant by a usual method. Usingthe isolated plasmid DNA as a template, and using DNAs having nucleotidesequences shown by SEQ ID NOS:33, 34, 35, 36 and 37 respectively asprimers, the nucleotide sequences of the inserted DNA fragments weredetermined with a DyeTerminator Cycle Sequencing Kit (produced byApplied Biosystem) and 373A sequencer (produced by Applied Biosystem),and the plasmid in which the nucleotide sequence shown by SEQ ID NO:44was inserted between SalI-BamHI site of pRI109, was named pSYN2-39.

The nucleotide sequence shown by SEQ ID NO:44 contained the nucleotidesequence which encodes the protein having the amino acid sequence shownby SEQ ID NO:45.

EXAMPLE 5 Introduction of the Plasmid into the C. glutamicum ATCC13032Strain and Evaluation of Activity

ATCC13032 strain was inoculated in a test tube containing 8 ml of brothmedium [20 g/l normal broth medium (produced by Kyokuto PharmaceuticalIndustry, Co. Ltd), 5 g/l Bacto Yeast Extract (produced by Difco)] andcultured with shaking 30° C. overnight. Subsequently, 5 ml of cellscultured overnight were inoculated in a 2 L Erienmeyer flask (bearing abuffle(s)) containing 250 ml of broth medium and cultured with shakingfor 4 hours at 30° C. The obtained culture solution was centrifuged toprecipitate the cells. After removing the supernatant, the cells weresuspended in 30 ml of ice-cold EPB [250 mmol/l Sucrose, 15% (v/v)glycerol], and centrifuged to be precipitated. Similarly, the cells wereresuspended in EPB and centrifuged to be separated, and then the cellswere suspended in 2 ml of EPB. The obtained cell suspension was pouredinto 0.5 ml tubes by 0.1 ml each, and was quickly frozen with dry ice toobtain the cell suspension for transformation. The obtained cells werestored at a temperature below −80° C.

0.1 ml of the frozen cell suspension for transformation was dissolved onice, retained for 10 minutes at 43.5° C., and transferred onto ice.After 2 μl of aqueous solution containing approximately 2 μg pRI109 DNAwas added, the cell suspension was transferred to the previously iced E.coli GenePulser cuvet (produced by BioRad), and then the DNA wasintroduced into cells under conditions of 25 μF, 200Ω and 1.5 kV byelectroporation using GenePulser (produced by BioRad). Immediately afterelectroporation, total amount of the cell suspension was moved to a 15ml-test tube containing 1 ml of broth medium, and cultured with shakingfor 1 hour at 30° C.

The obtained culture solution was centrifuged for 10 minutes at 3,500rpm to precipitate the cells. After removing the supernatant, the cellswere suspended with addition of 0.1 ml broth medium, then the suspensionwas applied to a broth agar medium [which was solidified with 2% DifcoAgar] containing 20 μg/ml kanamycin and cultured for 2 days at 30° C. toobtain the transformant.

Thus, C. glutamicum ATCC13032 strain having pRI109 was obtained.

As in the above, C. glutamicum ATCC13032 strains having each plasmid,pRIyjiB, pSYN2-72, pSYN2-39 were obtained.

The obtained transformants were inoculated in test tubes which contain 3ml of broth media containing 100 μg/ml kanamycin, and cultured withshaking for 24 hours at 30° C. The culture (0.2 ml) was inoculated in atest tube containing 2 ml of LMC medium [in which separately sterilizedGlucose, MgSO₄, FeSO₄, MnSO₄ were added to a pre-LMC medium sterilizedin a autoclave (NH₄Cl 1 g/l, KH₂PO₄ 1 g/l, K₂HPO₄ 3 g/l, Difco YeastExtract 0.2 g/l, Urea 1 g/l, Biotin 0.05 mg/l, Thiamin 0.5 mg/l, CornSteep Liquor 10 g/l; pH7.2) to the final concentration of 30 g/l, 0.1g/l, 2 mg/l and 2 mg/l, respectively] wherein the medium contains 100μg/ml kanamycin, and cultured with shaking for 5 hours at 30° C. Thecompound (VII-a) (wherein R is Na) was added thereto to the finalconcentration of 300 mg/l, and the mixture was reacted with shaking for16 hours at 30° C.

0.5 ml of the reaction solution was moved to a 1.5 ml tube, andcentrifuged for 2 minutes at 15,000 rpm to separate the cells. Theobtained supernatant was diluted 5 to 20 times with methanol andcentrifuged for 2 minutes at 15,000 rpm, and then an aliquot thereof wasused for HPLC analysis as in Example 1 to detect and quantify thecompound (VIII-a) (wherein R¹ is Na). The concentration of the compound(VIII-a) in the reaction solution calculated based on the quantificationresult, is shown in Table 4.

TABLE 4 Plasmid Compound (VIII-a) (mg/l) pRI109 0.3 pSYN2-72 30 pRIyjiB61 pSYN2-39 104

EXAMPLE 6 Introduction of the Plasmid into Coryne-Form Bacteria andEvaluation of Activity

pRIyjiB DNA obtained in Example 4 was introduced into C. callunaeATCC15991, C. ammoniagenes ATCC6872 and B. flavum ATCC14067 in the samemanner as in the transformation of ATCC13032 strain described in Example5, and transformants were obtained from each strain.

The obtained transformants were respectively inoculated on 3 ml of brothmedia in test tubes containing 100 μg/ml kanamycin, and cultured withshaking for 24 hours at 30° C. The culture (0.5 ml) was transferred to atest tube containing 5 ml TB medium [in which 14 g of Bacto Trypton(produced by Difco) and 24 g of Bacto Yeast Extract (produced by Difco)were dissolved in 900 ml of water and sterilized in an autoclave, towhich 100 ml PB [KH2PO₄ 23.1 g/l, K₂HPO₄ 125.1 g/l] separatelysterilized in an autoclave was added] wherein the medium contains 100μg/ml kanamycin and 10 g/l Glucose, and cultured with shaking for 5hours at 30° C. The culture (1 ml) was transferred to an assist tube(produced by ASSIST), and compound (VII-a) (wherein R is Na) was addedthereto to the final concentration of 300 mg/l, and the mixture wasreacted with shaking for 16 hours at 30° C.

Upon completion of reaction, compound (VIII-a) (wherein R¹ is Na) in theculture was detected and quantified in the method as in Example 2. Theconcentration of compound (VIII-a) in the culture calculated based onthe quantification results, is shown in Table 5.

TABLE 5 Compound (VIII-a) Host Cell Plasmid (mg/l) C. callunae ATCC15991(KY3510) pRIyjiB 22 C. ammoniagenes ATCC6872 (KY3454) pRIyjiB 12 B.flavum ATCC14067 (KY10122) pRIyjiB 23

INDUSTRIAL APPLICABILITY

The present invention enables efficient production of a DNA encoding anovel hydroxylase and a compound inhibiting hydroxymethylglutaryl CoA(HMG-CoA) reductase and has an action of reducing serum cholesterol.

Free Text of Sequence Listing SEQ ID NO:3 synthetic DNA SEQ ID NO:4synthetic DNA SEQ ID NO:5 synthetic DNA SEQ ID NO:6 synthetic DNA SEQ IDNO:7 synthetic DNA SEQ ID NO:8 synthetic DNA SEQ ID NO:9 synthetic DNASEQ ID NO:10 synthetic DNA SEQ ID NO:11 synthetic DNA SEQ ID NO:12synthetic DNA SEQ ID NO:13 synthetic DNA SEQ ID NO:14 synthetic DNA SEQID NO:15 synthetic DNA SEQ ID NO:16 synthetic DNA SEQ ID NO:17 syntheticDNA SEQ ID NO:18 synthetic DNA SEQ ID NO:19 synthetic DNA SEQ ID NO:20synthetic DNA SEQ ID NO:21 synthetic DNA SEQ ID NO:22 synthetic DNA SEQID NO:23 synthetic DNA SEQ ID NO:24 synthetic DNA SEQ ID NO:25 syntheticDNA SEQ ID NO:26 synthetic DNA SEQ ID NO:27 synthetic DNA SEQ ID NO:28synthetic DNA SEQ ID NO:29 synthetic DNA SEQ ID NO:30 synthetic DNA SEQID NO:31 synthetic DNA SEQ ID NO:32 synthetic DNA SEQ ID NO:33 syntheticDNA SEQ ID NO:34 synthetic DNA SEQ ID NO:35 synthetic DNA SEQ ID NO:36synthetic DNA SEQ ID NO:37 synthetic DNA SEQ ID NO:38 synthetic DNA SEQID NO:39 synthetic DNA SEQ ID NO:40 synthetic DNA

1. An isolated DNA having the nucleotide sequence shown by SEQ ID NO: 2.2. A recombinant DNA vector comprising the DNA according to claim
 1. 3.An isolated transformed cell obtained by introducing the recombinant DNAvector according to claim 2 into a host cell.
 4. The transformed cellaccording to claim 3 wherein the transformed cell belongs to amicroorganism selected from the genera Escherichia, Bacillus,Corynebacterium, and Streptomyces.
 5. The transformed cell according toclaim 3 wherein the transformed cell belongs to microorganism selectedfrom Escherichia coli, Bacillus subtilis, Bacillus megaterium,Corynebacterium glutamicum, Corynebacterium ammoniagenes,Corynebacterium callunae and Streptomyces lividans.
 6. A process forproducing compound (II-a) or compound (II-b), wherein The transformedcell according to claim 3, a culture of the transformed cell, or atreated product of the culture is used as an enzyme source, and theprocess comprises: allowing compound (I-a) or compound (I-b) to exist inan aqueous medium in the presence of the enzyme source; allowingcompound (II-a) or compound (II-b) to be produced and accumulated insaid aqueous medium; and collecting compound (II-a) or compound (II-b)from said aqueous medium wherein, the compound (I-a) is a compoundrepresented by the formula (I-a):

the compound (I-b) is a lactone form of compound (I-a) and isrepresented by the formula (I-b):

the compound (II-a) is a compound represented by the formula (II-a):

the compound (II-b) is a lactone form of compound (II-a) and isrepresented by the formula (II-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl; andwherein the treated product of the culture of the transformed cell is atreated product selected from cultured cells, dried cells, freeze-driedcells, cells treated with a surfactant, cells treated with an enzyme,cells treated by ultrasonication, cells treated by mechanical milling,cells treated by solvent, a protein fraction of a cell, and immobilizedcells.
 7. A process for producing compound (IV-a) or compound (IV-b),wherein the transformed cell according to claim 3, a culture of thetransformed cell, or a treated product of the culture is used as anenzyme source, and the process comprises: allowing compound (III-a) orcompound (III-b) to exist in an aqueous medium in the presence of theenzyme source; allowing compound (IV-a) or compound (IV-b) to beproduced and accumulated in said aqueous medium; and collecting compound(IV-a) or compound (IV-b) from said aqueous medium wherein, the compound(III-a) is a compound represented by the formula (III-a):

the compound (III-b) is a lactone form of compound (III-a) and isrepresented by the formula (III-b):

the compound (IV-a) is a compound represented by the formula (IV-a):

the compound (IV-b) is a lactone form of compound (IV-a) and isrepresented by the formula (IV-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl; andwherein the treated product of the culture of the transformed cell is atreated product selected from cultured cells, dried cells, freeze-driedcells, cells treated with a surfactant, cells treated with an enzyme,cells treated by ultrasonication, cells treated by mechanical milling,cells treated by solvent, a protein fraction of a cell, and immobilizedcells.
 8. A process for producing compound (VI-a) or compound (VI-b),wherein the transformed cell according to claim 3, a culture of thetransformed cell, or a treated product of the culture is used as anenzyme source, and the process comprises: allowing compound (V-a) orcompound (V-b) to exist in an aqueous medium in the presence of theenzyme source; allowing compound (VI-a) or compound (VI-b) to beproduced and accumulated in said aqueous medium; and collecting compound(VI-a) or compound (VI-b) from said aqueous medium wherein, the compound(V-a) is a compound represented by the formula (V-a):

the compound (V-b) is a lactone form of compound (V-a) and isrepresented by the formula (V-b):

the compound (VI-a) is a compound represented by the formula (VI-a):

the compound (VI-b) is a lactone form of compound (VI-a) and isrepresented by the formula (VI-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal; and wherein the treated productof the culture of the transformed cell is a treated product selectedfrom cultured cells, dried cells, freeze-dried cells, cells treated witha surfactant, cells treated with an enzyme, cells treated byultrasonication, cells treated by mechanical milling, cells treated bysolvent, a protein fraction of a cell, and immobilized cells.
 9. Aprocess for producing compound (VIII-a) or compound (VIII-b), whereinthe transformed cell according to claim 3, a culture of the transformedcell, or a treated product of the culture is used as an enzyme source,and the process comprises: allowing compound (VII-a) or compound (VII-b)to exist in an aqueous medium in the presence of the enzyme source;allowing compound (VII-a) or compound (VII-b) to be produced andaccumulated in said aqueous medium; and collecting compound (VIII-a) orcompound (VIII-b) from said aqueous medium wherein, the compound (VII-a)is a compound represented by the formula (VII-a):

the compound (VII-b) is a lactone form of compound (VII-a) and isrepresented by the formula (VII-b):

the compound (VIII-a) is a compound represented by the formula (VIII-a):

the compound (VII-b) is a lactone form of compound (VIII-a) and isrepresented by the formula (VIII-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal; and wherein the treated productof the culture of the transformed cell is a treated product selectedfrom cultured cells, dried cells, freeze-dried cells, cells treated witha surfactant, cells treated with an enzyme, cells treated byultrasonication, cells treated by mechanical milling, cells treated bysolvent, a protein fraction of a cell, and immobilized cells.
 10. Theprocess according to claim 6, wherein the compound (II-b) is obtained byforming a lactone from compound (II-a), the compound (II-a) is acompound represented by the formula (II-a):

the compound (II-b) is a lactone form of compound (II-a) and isrepresented by the formula (II-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl.
 11. Theprocess according to claim 6, wherein the compound (II-a) is obtained byopening the lactone ring of compound (II-b), the compound (II-a) is acompound represented by the formula (II-a):

the compound (II-b) is a lactone form of compound (II-a) and isrepresented by the formula (II-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl.
 12. Theprocess according to claim 7, wherein the compound (IV-b) is obtained byforming a lactone from compound (IV-a), the compound (IV-a) is acompound represented by the formula (IV-a):

the compound (IV-b) is a lactone form of compound (IV-a) and isrepresented by the formula (IV-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl.
 13. Theprocess according to claim 7, wherein the compound (IV-a) is obtained byopening the lactone ring of compound (IV-b), the compound (IV-a) is acompound represented by the formula (IV-a):

the compound (IV-b) is a lactone form of compound (IV-a) and isrepresented by the formula (IV-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl.
 14. Theprocess according to claim 8, wherein the compound (VI-b) is obtained byforming a lactone from compound (VI-a), the compound (IV-a) is acompound represented by the formula (IV-a):

the compound (IV-b) is a lactone form of compound (IV-a) and isrepresented by the formula (IV-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal, and R² represents a substitutedor unsubstituted alkyl, or a substituted or unsubstituted aryl.
 15. Theprocess according to claim 8, wherein the compound (VI-a) is obtained byopening the lactone ring of compound (VI-b), the compound (VI-a) is acompound represented by the formula (VI-a):

the compound (VI-b) is a lactone form of compound (VI-a) and isrepresented by the formula (VI-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal.
 16. The process according toclaim 9, wherein the compound (VIII-b) is obtained by forming a lactonefrom compound (VIII-a), the compound (VIII-a) is a compound representedby the formula (VIII-a):

the compound (VIII-b) is a lactone form of compound (VIII-a) and isrepresented by the formula (VIII-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal.
 17. The process according toclaim 9, wherein the compound (VIII-a) is obtained by opening thelactone ring of compound (VIII-b), the compound (VIII-a) is a compoundrepresented by the formula (VIII-a):

the compound (VIII-b) is a lactone form of compound (VIII-a) and isrepresented by the formula (VIII-b):

and wherein R¹ represents a hydrogen atom, a substituted orunsubstituted alkyl, or an alkali metal.
 18. A process for producing aprotein, which is derived from a microorganism belonging to the genusBacillus, and has an activity of producing compound (II-a) or compound(II-b) from compound (I-a) or compound (I-b). wherein the compound (I-a)is a compound represented by the formula (I-a):

the compound (I-b) is a lactone form of compound (I-a) and isrepresented by the formula (I-b):

the compound (II-a) is a compound represented by the formula (II-a):

the compound (II-b) is a lactone form of compound (II-a) and isrepresented by the formula (II-b):

wherein R¹ represents a hydrogen atom, a substituted or unsubstitutedalkyl, or an alkali metal, and R² represents a substituted orunsubstituted alkyl, or a substituted or unsubstituted aryl, whichcomprises culturing a transformed cell obtained by introducing arecombinant DNA vector comprising the DNA having the nucleotide sequenceshown by SEQ ID NO: 2 in a medium; producing and accumulating theprotein in the culture; and collecting the protein from said culture.